The effect of hydrogen peroxide on the cyclin D expression in fibroblasts

Activation of mitogen-activated protein (MAP) kinase is essential for cyclin D1 expression and provides a link between mitogenic signalling and cell cycle progression. Hydrogen peroxide (H₂ O₂ ) activates MAP kinase; however, it is not known whether this leads to cyclin D expression. Sustained expression of cyclin D1 and D2 was observed when Her14 fibroblasts were incu-bated with 3 mM or higher H₂ O₂ concentrations. Similar results were obtained when cells were incubated in the presence of serum (FCS). However, the sustained expres-complex sion of cyclin D1 and D2 upon H₂ O₂ treatment was not due to the MAP kinase pathway, because MAP kinase kinase inhibitors did not inhibit cyclin D expression. Furthermore, cyclin D1 and D2 levels remained constant even after addition of a protein synthesis inhibitor, indicating that the effect of H₂ O₂ was not due to induction of protein synthesis. These results indicate that H₂ O₂ reversibly inhibits the ubiquitin-proteasome dependent degra-dation of cyclin D1 and D2, probably by transiently in-hibiting ubiquitination and/or the proteasome. Received 12 March 2001; received after revision 5 April 2001; accepted 9 April 2001     

D2A sequence of the urokinase receptor induces cell growth through αvβ3 integrin and EGFR

The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvβ3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.     

Wip1 protects hydrogen peroxide-induced colonic epithelial barrier dysfunction

Tight junctions (TJs) create a paracellular permeability barrier. Although reactive oxygen species have been implicated as mediators of inflammation in inflammatory bowel diseases, their influence on the function of colonic epithelial TJs remains unknown. Oxidative stress-mediated colonic epithelial permeability was significantly attenuated by a p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Although the amount of TJ proteins was not altered, hydrogen peroxide (H₂O₂) changed the localization of claudin-4 protein from an NP-40 insoluble fraction to a soluble fraction and from an apical TJ to lateral membrane. The p38 MAP kinase inactivator Wip1 significantly attenuated phosphorylation of p38 MAP kinase, and oxidative stress mediated permeability. H₂O₂-induced changes in claudin-4 localization were abolished by SB203580 pretreatment as well as Wip1-expressing adenovirus infection. This is the first study to demonstrate that exogenous Wip1 functions to protect oxidative stress-mediated colonic mucosal permeability and that H₂O₂-induced claudin-4 dislocalization is abolished by Wip1. Received 14 June 2007; received after revision 8 October 2007; accepted 8 October 2007     

Cytoprotective effects of acidosis via heat shock protein HSP27 against the anticancer drug doxorubicin

Drug resistance continues to be a stumbling block in achieving better cure rates in several cancers. Doxorubicin is commonly used in treatment of a wide range of cancers. The aim of this study was to look into the mechanisms of how low ambient pH may contribute to down-regulation of apoptotic pathways in a gastric tumour cell line. Low pH culture conditions were found to dramatically prolong cell survival after doxorubicin treatment, an effect that was in part reversed by co-incubation with the specific p38 mitoge-activated protein kinase (MAP kinase) inhibitor SB203580, only mildly inhibited by blockade of the multi-drug resistance 1 (MDR1) transporter, but completely abolished by siRNA-mediated knockdown of the heat shock protein 27 (HSP27). In conclusion, acidic pH causes less accumulation of cytotoxic drug in the nucleus of adeno gastric carcinoma (AGS) cells and HSP27-dependent decrease in FasR-mediated gastric epithelial tumour cell apoptosis.     

The multi-targeted kinase inhibitor sorafenib inhibits human cytomegalovirus replication

Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised individuals. Here, non-toxic concentrations of the anti-cancer kinase inhibitor sorafenib were shown to inhibit replication of different HCMV strains (including a ganciclovir-resistant strain) in different cell types. In contrast to established anti-HCMV drugs, sorafenib inhibited HCMV major immediate early promoter activity and HCMV immediate early antigen (IEA) expression. Sorafenib is known to inhibit Raf. Comparison of sorafenib with the MEK inhibitor U0126 suggested that sorafenib inhibits HCMV IEA expression through inhibition of Raf but independently of signaling through the Raf downstream kinase MEK 1/2. In concordance, siRNA-mediated depletion of Raf but not of MEK-reduced IEA expression. In conclusion, sorafenib diminished HCMV replication in clinically relevant concentrations and inhibited HCMV IEA expression, a pathophysiologically relevant event that is not affected by established anti-HCMV drugs. Moreover, we demonstrated for the first time that Raf activation is involved in HCMV IEA expression.     

Regulation of HERG (KCNH2) potassium channel surface expression by diacylglycerol

The HERG (KCNH2) channel is a voltage-sensitive potassium channel mainly expressed in cardiac tissue, but has also been identified in other tissues like neuronal and smooth muscle tissue, and in various tumours and tumour cell lines. The function of HERG has been extensively studied, but it is still not clear what mechanisms regulate the surface expression of the channel. In the present report, using human embryonic kidney cells stably expressing HERG, we show that diacylglycerol potently inhibits the HERG current. This is mediated by a protein kinase C-evoked endocytosis of the channel protein, and is dependent on the dynein–dynamin complex. The HERG protein was found to be located only in early endosomes and not lysosomes. Thus, diacylglycerol is an important lipid participating in the regulation of HERG surface expression and function.     

Inhibition of invasion and intraerythrocytic development ofPlasmodium falciparum by kinase inhibitors

We have examined the effects of seven protein kinase inhibitors (staurosporine, genistein, methyl 2,5-dihydroxycinnamate, tyrphostins B44 and B46, lavendustin A and R03) on the erythrocytic cycle of the malaria parasite,Plasmodium falciparum. One (staurosporine) strongly inhibits serine/threonine kinases, but the remainder all exhibit a strong preference for tyrosine kinases. We have been able to discriminate between effects on invasion and on intraerythrocytic development. All reagents impeded development of intraerythrocytic parasites, though at widely differing concentrations, from the sub-micromolar to the millimolar. Several inhibitors, including staurosporine, also reduced invasion. The phosphatase inhibitor, okadaic acid, had a strong inhibitory effect both on invasion and development. The regulation of malaria development by phosphorylation or dephosphorylation reactions at several points in the blood-stage cycle is implied.     

Signaling pathways between the plasma membrane and endoplasmic reticulum calcium stores

This review discusses multiple ways in which the endoplasmic reticulum participates in and is influenced by signal transduction pathways. The endoplasmic reticulum provides a Ca2+ store that can be mobilized either by calcium-induced calcium release or by the diffusible messenger inositol 1,4,5-trisphosphate. Depletion of endoplasmic reticulum Ca2+ stores provides a signal that activates surface membrane Ca2+ channels, a process known as capacitative calcium entry. Depletion of endoplasmic reticulum stores can also signal long-term cellular responses such as gene expression and programmed cell death or apoptosis. In addition to serving as a source of cellular signals, the endoplasmic reticulum is also functionally and structurally modified by the Ca2+ and protein kinase C pathways. Elevated cytoplasmic Ca2+ causes a rearrangement and fragmentation of endoplasmic reticulum membranes. Protein kinase C activation reduces the storage capacity of the endoplasmic reticulum Ca2+ pool. In some cell types, protein kinase C inhibits capacitative calcium entry. Protein kinase C activation also protects the endoplasmic reticulum from the structural effects of high cytoplasmic Ca2+. The emerging view is one of a complex network of pathways through which the endoplasmic reticulum and the Ca2+ and protein kinase C signaling pathways interact at various levels regulating cellular structure and function.     

Effects of cytochalasin D on the actin cytoskeleton: association of neoformed actin aggregates with proteins involved in signaling and endocytosis

Cytochalasin D (CD) has been extensively used for assessing the role of the actin cytoskeleton in different biological processes. However, effects of CD have not always been consistent and CD-treated cells have been found to contain irregular spots of F-actin. By transfecting MCF-7 cells with an actin-enhanced yellow fluorescent protein fusion protein we show that, in vivo, CD induces actin aggregation de novo, while simultaneously depolymerizing preexisting actin cytoskeletal components. We also show that CD-induced actin aggregates bind the F-actin-selective drug phalloidin and associate with proteins involved in cell signaling as well as with receptors and endosomal markers (active MAP kinases, paxillin, erbB2, transferrin, Rab-5), but not with clathrin, protein kinase A, protein tyrosine phosphatase 1B, or tubulin. Thus, CD induces new sites of actin aggregation that selectively associate with several important regulatory proteins. Failure of CD to interupt a biological process may therefore not prove that the process is independent of actin aggregation.     

The p38/MAPK pathway regulates microtubule polymerization through phosphorylation of MAP4 and Op18 in hypoxic cells

In both cardiomyocytes and HeLa cells, hypoxia (1% O₂) quickly leads to microtubule disruption, but little is known about how microtubule dynamics change during the early stages of hypoxia. We demonstrate that microtubule associated protein 4 (MAP4) phosphorylation increases while oncoprotein 18/stathmin (Op18) phosphorylation decreases after hypoxia, but their protein levels do not change. p38/MAPK activity increases quickly after hypoxia concomitant with MAP4 phosphorylation, and the activated p38/MAPK signaling leads to MAP4 phosphorylation and to Op18 dephosphorylation, both of which induce microtubule disruption. We confirmed the interaction between phospho-p38 and MAP4 using immunoprecipitation and found that SB203580, a p38/MAPK inhibitor, increases and MKK6(Glu) overexpression decreases hypoxic cell viability. Our results demonstrate that hypoxia induces microtubule depolymerization and decreased cell viability via the activation of the p38/MAPK signaling pathway and changes the phosphorylation levels of its downstream effectors, MAP4 and Op18.     

Differential protein expression in pancreatic islets after treatment with an imidazoline compound

The effects of an imidazoline compound (BL11282) on protein expression in rat pancreatic islets were investigated with a proteomic approach. The compound increases insulin release selectively at high glucose concentrations and is therefore of interest in type 2 diabetes. Whole cell extracts from isolated drug-treated and native pancreatic rat islets were compared after separation by 2-D gel electrophoresis. Differentially expressed proteins were identified by mass spectrometry; 15 proteins were selectively up-regulated and 7 selectively down-regulated in drug-treated islets. Of special interest among the differentially expressed proteins are those involved in protein folding (Hsp60, protein disulfide isomerase, calreticulin), Ca²⁺ binding (calgizzarin, calcyclin and annexin I) and metabolism or signalling (pyruvate kinase, alpha enolase and protein kinase C inhibitor 1). Received 19 March 2007; received after revision 11 April 2007; accepted 11 April 2007     

Forskolin inhibits tension development in detergent-treated cardiac muscle fibers

Summary In detergent-treated cardiac muscle fibers, forskolin, a potent activator of adenylate cyclases, inhibits tension development elicited with submaximal [Ca²⁺] and increases incorporation of³²P into troponin-I. A similar reduced tension development has been observed after treatment with cAMP or the catalytic subunit of the cAMP-dependent protein kinase. It is concluded that these fibers still contain much of the enzyme cascade involved in evoking a contractile response to \-adrenergic stimulation.Acknowledgment. The authors thank Dr H. Metzger, Hoechst AG, Frankfurt (FRG) for the gift of forskolin. The excellent technical assistance of Mrs Doris Eubler and the support by the Deutsche Forschungsgemeinschaft (SFB 90) are gratefully acknowledged.     

Action of juvenile hormone on the follicle cells ofRhodnius prolixus: Evidence for a novel regulatory mechanism involving protein kinase C

Summary Juvenile hormone (JH) is known to act on the membranes of the follicle cells ofRhodnius, activating a specific Na⁺, K⁺-ATPase. This leads to a decrease in volume of the cells and the appearance of spaces between them (patency). The addition of an inhibitor of protein kinase C, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), to the medium in vitro inhibits the action of JH on the follicle cells. PDBU (phorbol-12,13-dibutyrate) mimics the action of JH in vitro and the response of the follicle cells to, PDBU is blocked by ouabain. It is concluded that the activation of protein kinase C is a required step in the chain of events leading to activation of the JH-dependent ATPase and set in train by the binding of JH to the membrane.     

Evaluation of signal transduction pathways mediating the nuclear exclusion of the androgen receptor by melatonin

The intracellular signaling pathways mediating the nuclear exclusion of the androgen receptor (AR) by melatonin were evaluated in PC3 cells stably transfected with the AR. The melatonin-induced nuclear exclusion of the AR by melatonin (100 nM, 3 h) was blocked by LY 83583 (an inhibitor of guanylyl cyclases). 8-Bromo-cGMP (a cell-permeable cGMP analog), mimicked the effect of melatonin, as did ionomycin (a calcium ionophore) and PMA [an activator of protein kinase C (PKC)], and their effects were blocked by GF-109203X (a selective PKC inhibitor). BAPTA (an intracellular calcium chelator) blocked the effects of melatonin and 8-bromo-cGMP but not of PMA. Inhibition or activation of the protein kinase A pathway did not affect basal or melatonin-mediated AR localization. We conclude that the melatonin-mediated rise in cGMP elicits AR nuclear exclusion via a pathway involving increased intracellular calcium and PKC activation. These results define a novel signaling pathway that regulates AR localization and androgen responses in target cells. Received 31 July 2001; received after revision 18 September 2001; accepted 30 October 2001     

Calyculin A increases voltage-dependent inward current in smooth muscle cells isolated from guinea pig taenia coli.

The effects of a potent phosphatase inhibitor, calyculin A (CL-A), on inward currents in guinea pig taenia coli smooth muscle cells were examined. CL-A increased the inward current, and this effect of CL-A was inhibited by a protein kinase C inhibitor, H-7, and by nifedipine. Phorbol 12,13-dibutyrate, an activator of protein kinase C, also increased the inward current and this effect was antagonized by H-7. These results suggest that in guinea pig taenia coli smooth muscle cells CL-A may facilitate the opening of the L-type Ca2+ channels through the protein kinase C-dependent phosphorylation system.